Non-invasive Ultrasound Mechanotherapy Sensitizes Prostate Tumors to TRAIL-Induced Apoptosis

Introduction: The King Lab has previously demonstrated how fluid shear stress (FSS) sensitizes breast, colon, and prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through Piezo1 activation of the intrinsic apoptotic pathway [1]. Piezo1 is a mechanosensitive ion channel (MSC) that is activated by external stimuli and cancer cells recognize and respond to these signals, leading to calcium influx to promote pro-apoptotic pathways [1,2]. We aimed to explore the biochemical response that occurs in prostate cancer (PC) when treated with therapeutic, focal ultrasound (FUS) combined with TRAIL. TRAIL binds to death receptors 4 and 5 (DR4/5) on cancer cells, while sparing healthy cells, however soluble TRAIL on its own lacks efficacy in humans to be used as a cancer therapy.

Materials and

Methods: Sensitization of TRAIL-mediated apoptosis via FUS was examined in weakly metastatic LNCaP cells and highly metastatic PC3 cells. The FUS parameters used in vitro and in vivo are listed in Figure 1A. The FUS system is depicted in Fig. 1B. For the results shown in Figures 1D-H, PC3 cells were treated with TRAIL (200 ng/mL) immediately before FUS treatment. A Piezo1 inhibitor, GsMTx-4 (10 µM) (Fig. 1G-H), and a calcium chelator, EGTA (2 mM), were used to confirm MSC activation during FUS+TRAIL treatment. Viability and apoptosis were assessed using the Annexin-V/ propidium iodide (AV/PI) assay and mitochondrial depolarization was measured using a JC-1 assay via flow cytometry, both 24 h after treatment. An in vivo study used eight-week-old, male NU/NU mice (Fig. 1I-J) bearing subcutaneous PC3 tumors inoculated on each flank. Soluble TRAIL (2 mg/kg) was delivered intratumorally, followed by a single FUS treatment (10 min/tumor), using the same pulse repetition frequency, duty cycle, transducer and burst duration as in Figure 1A at 944 kPa. Tumors were processed by the Translational Pathology Core at Vanderbilt for immunohistochemistry (IHC) analysis upon resection.

Results, Conclusions, and Discussions: Combination treatment delineated a pressure-dependent increase in TRAIL-mediated apoptosis and mitochondrial depolarization for PC3 cells (Fig. 1D-F), complemented by an increase in TRAIL sensitization. Treatment of PC3 cells with GsMTx-4 (Fig. 1G-H) and EGTA reduced TRAIL sensitization upon combination treatment, indicating activation of pro-apoptotic pathways in response to FUS treatment.

The in vivo study demonstrates, for the first time, the use of FUS to decrease tumor burden when applied with soluble TRAIL (Fig. 1I-J), evident by significantly decreased tumor volume for the combination treatment. IHC analysis of the tumors revealed upregulated Piezo1 expression for the FUS+TRAIL-treated tumors compared to FUS- or TRAIL-only therapy. Enhanced cleaved caspase-3 and TUNEL staining, both markers of cell death, was also observed for the FUS+TRAIL combination therapy, further supporting successful tumor cell ablation following treatment.

TRAIL and FUS combination therapies can provide an alternative, non-invasive mechanobiology-based therapeutic, which employs technology already widely available. For these experiments, the maximum pressure examined was 1993 kPa, and most experiments were performed at or below 944 kPa, at which the mechanical index (MI) is 0.9. An MI of 1.9 is the limit considered safe by the FDA before any major cavitation is expected to occur; higher values of MI increase the probability of mechanical damage [3]. This is critical considering that the prostate is located near neurovascular bundles that risk being damaged during invasive treatments options [4]. Future work will apply these studies to additional cancer types, test alternate FUS parameters, and determine if additional MSCs are being activated and contributing to the TRAIL-sensitization of cancer cells via FUS exposure.

Acknowledgements (Optional): This research was funded by the NSF-GRFP.

References: [1] JM Hope, Cell Death & Dis, 2019, 10, 1-15; [2] JA Dombroski, Cells, 2021, 10, 1-14; [3] Şen, T., Tüfekçioğlu, O. & Koza, Y. Anatol J Cardiol, 2015, 15, 334–336; [4] Y Wu; Nature Biomed. Engr, 2021, 5, 1336-47.